Cell movement in response to specific stimuli is observed to occur in prokaryotes and eukaryotes (Doetsch R N and Seymour W F., 1970; Bailey G B et al., 1985). Cell movement seen in these organisms has been classified into three types; chemotaxis or the movement of cells along a gradient towards an increasing concentration of a chemical; negative chemotaxis which has been defined as the movement down a gradient of a chemical stimulus and chemokinesis or the increased random movement of cells induced by a chemical agent. The receptors and signal transduction pathways for the actions of specific chemotactically active compounds have been extensively defined in prokaryotic cells. Study of E. Coli chemotaxis has revealed that a chemical which attracts the bacteria at some concentrations and conditions may also act as a negative chemotactic chemical or chemorepellent at others (Tsang N et al., 1973; Repaske D and Adler J. 1981; Tisa L S and Adler J., 1995; Taylor B L and Johnson M S., 1998).
Chemotaxis and chemokinesis have been observed to occur in mammalian cells (McCutcheon M W, Wartman W and H M Dixon, 1934; Lotz M and H Harris; 1956; Boyden S V 1962) in response to the class of proteins, called chemokines (Ward S G and Westwick J; 1998; Kim C H et al., 1998; Baggiolini M, 1998; Farber J M; 1997).
Chemokines induce cell locomotion by signaling through G-protein coupled receptors (Wells T N et al., 1998). The membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane α-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
G-protein coupled receptors have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1 receptor, rhodopsins, odorant, cytomegalovirus receptors, etc.
Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction. Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors. Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy-terminus. For several G-protein coupled receptors, such as the β-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
For some receptors, the ligand binding sites of G-protein coupled receptors are believed to comprise a hydrophilic socket formed by several G-protein coupled receptors transmembrane domains, which socket is surrounded by hydrophobic residues of the G-protein coupled receptors. The hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form the polar ligand binding site. TM3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as including the TM3 aspartate residue. Additionally, TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding.
G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc Rev, 1989, 10:317-331). Different G-protein a-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors have been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host. Chemokine-induced cell chemotaxis and chemokinesis are thought to be mediated via a Gαi-linked signal transduction pathway and can be blocked by pertussis toxin (PTX) (Luster A D, 1998; Baggiolini, 1998).
The chemokine, SDF-1α, causes immigration of subpopulations of leukocytes into sites of inflammation (Aiuti A et al. 1997; Bleul C C et al. 1996; Bleul C C et al., 1996; Oberlin E et al., 1996). Furthermore, mice engineered to be deficient in SDF-1α or its receptor, CXCR-4, have abnormal development of hematopoietic tissues and B-cells manifesting a failure of fetal liver stem cells to migrate to bone marrow (Friedland J S, 1995; Tan J and Thestrup-Pedersen K, 1995; Corrigan C J and Kay A B, 1996; Qing M, et al, 1998; Ward S G et al. 1998).
Although chemotaxis and chemokinesis have been defined in cell subpopulations in mammals, negative chemotaxis of peripheral blood cells from mammals has only been observed in response to non-specific stimuli such as cell lysates or tumor tissue fragments (Jochims, 1927; McCutcheon et al. 1939; Bessis M and Burte B., 1965; Noble and Bentley, 1981) and reports have been very limited. The precise mechanism of negative chemotaxis in higher eukaryotic cell subpopulations, including the definition of specific stimuli and signal transduction pathways of negative chemotaxis has not been defined. Furthermore, although chemoattractants had been shown to serve as repellents in prokaryotic systems, no analogous system of a dual action chemoattractant/chemorepellent compound has been identified in higher eukaryotes.